Bioaccumulation and in vivo tracking of radiolabeled 4-nonylphenol in mice

4-Nonylphenol (4NP) is concerning due to its growing presence and endocrine-disrupting nature, raising concerns about its impact on health. In this study 124I-labeled 4NP was synthesized for in vivo tracing. Positron emission tomography imaging and biodistribution studies showed significant accumulation in various tissues after oral or intraperitoneal administration, emphasizing its intricate distribution and potential long-term effects, crucial for future risk assessments.

The 1 H and 13 C nuclear magnetic resonance (NMR) spectra were obtained using Bruker Avance III HD NMR spectrometer at 600MHz.DMSO-d 6 was used as the solvent.The radioactive and nonradioactive 4NP analogs were analyzed and purified using the Waters Alliance semipreparative high-performance liquid chromatography (HPLC) system equipped with gamma detector and XTerra Prep RP 18 columns (10 × 250 mm, 10.0μm).The solvent compositions A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile) were used for the HPLC experiments.The method involved a linear gradient approach with a flow rate of 4 mL/min.The gradient was programmed as follows: 0-3 min (95% A/5% B), 3-20 min (20% A/80% B), 20-30 min (5% A/95% B), 30-40 min (95% A/5% B), and 40-45 min (5% A/95% B).The in vitro stability of the radiolabeled compound was monitored using an Eckert & Ziegler radio-thin-layer-chromatography (TLC) scanner (model: AR-2000) and HPLC system.The biodistribution study was performed on 6-week-old ICR mice, supplied by DooYeol Biotech (Seoul, Republic of Korea).The radioactivity accumulated in each organ was quantified using the PerkinElmer automatic gamma counter (model: 2480 automatic gamma counter).PET images were acquired using the Mediso nanoScan PET system.All animal experiments were approved by the KIRAMS Committee for Animal Welfare and were conducted in full compliance with the guidelines outlined in the Korean Animal Protection Law.

Production of 124 I
Radioiodine ( 124 I, 370MBq/100μL), supplied by KIRAMS, Seoul Republic of Korea, was provided in a 0.1N solution of sodium hydroxide.

Synthesis of radioiodinated 4NP ( 124 I-labeled 4NP)
A solution of 4NP (2mg, 0.009 mmol) in DMSO (200μL) was prepared.Sequentially, acetic acid (50μL) and chloramine-T (1.5mg, 15μL) were added to this solution.To initiate the reaction sequence for radioiodination, a solution of [ 124 I]NaI (185 MBq) in 0.1 M NaOH (150μL) was introduced into the reaction mixture, which was then incubated at room temperature for 30 min.
To terminate the reaction, a solution of sodium metabisulfite solution (20mg, 20μL) was added.
The crude reaction product was subjected to purification using HPLC.The purified product was diluted with 10 mL of deionized water and loaded into a C18 Sep-Pak cartridge, which was previously conditioned with ethanol (6 mL) followed by deionized water (12 mL).Subsequent elution of the trapped product was accomplished using absolute ethanol (500 μL), and the eluted solution was then diluted with saline in preparation for further in vitro and in vivo experiments.

In vitro stability of 124 I-labeled 4NP
For assessment of in vitro stability, a sample of purified 124 I-labeled 4NP (740 kBq, 10μL) was incubated in 500μL of mouse serum (90%) and 0.1M HCl or saline, at room temperature for 48 h.Stability analyses were conducted at specific intervals (1, 6, 12, 24, 36, and 48h) using either a radio-TLC scanner or a HPLC system.Each experiment was replicated thrice to ensure reliability and consistency of results

PET imaging of 124 I-labeled 4NP
Comprehensive in vivo studies were carried out on a state-of-the-art PET system.The 124 Ilabeled 4NP, at a concentration of 5.55 MBq/100 μL in saline medium, was introduced into mice via the oral or I.P. route.Subsequent PET imaging was conducted under 2% isoflurane at multiple temporal intervals.

Biodistribution study of 124 I-labeled 4NP
For the biodistribution analyses, a 124 I-labeled 4NP concentration of 740 kBq/100 μL in saline matrix was maintained.The radiolabeled compound was introduced systemically via oral or I.P. administration.At specified intervals, four mice per time point were euthanized, and their respective organs and blood samples were harvested.The radiotracer accumulation within each organ was quantified using an automated gamma counter, and the outcomes were expressed as a percentage of the injected dose per gram (%ID/g) of the organ.

Table S1 .
The biodistribution data of 124 I-labeled 4NP after oral administration (n=4), data represented as %ID/g of organ or blood along with standard deviation values

Table S2 .
The biodistribution data of 124 I-labeled 4NP after intraperitoneal injection (n=4), data represented as %ID/g of organ or blood along with standard deviation values